Tissue Determinants of Human NK Cell Development, Function, and Residence

Abstract

Immune responses in diverse tissue sites are critical for protective immunity and homeostasis. Here, we investigate how tissue localization regulates the development and function of human natural killer (NK) cells, innate lymphocytes important for anti-viral and tumor immunity. Integrating high-dimensional analysis of NK cells from blood, lymphoid organs, and mucosal tissue sites from 60 individuals, we identify tissue-specific patterns of NK cell subset distribution, maturation, and function maintained across age and between individuals. Mature and terminally differentiated NK cells with enhanced effector function predominate in blood, bone marrow, spleen, and lungs and exhibit shared transcriptional programs across sites. By contrast, precursor and immature NK cells with reduced effector capacity populate lymph nodes and intestines and exhibit tissue-resident signatures and site-specific adaptations. Together, our results reveal anatomic control of NK cell development and maintenance as tissue-resident populations, whereas mature, terminally differentiated subsets mediate immunosurveillance through diverse peripheral sites. VIDEO ABSTRACT.

Keywords: NK cells; cytotoxicity; human immunology; immune development; immunity of aging; innate immunity; lymphoid tissues; mucosal immunity; systems immunology; tissue residence.

Figure 1.. Human NK Cell Subset Distribution Is a Function of Tissue Site

(A) Schematic diagram of the human tissues obtained and experimental workflow presented in this study. (B) Gating strategy used for identification of NK cells and subsets by flow cytometry for analysis and sorting. (C) NK cell distribution in different human tissue compartments depicted in representative flow cytometry plots (left, D344) showing NK cell (CD45⁺CD14⁻19⁻CD3⁻CD56⁺) frequency, and boxplots of compiled data from 18–55 donors (right). See also Figure S1A, left panel. (D) Distribution of CD56^brightCD16⁻ (light orange) and CD56^dimCD16⁺ (blue) NK cell subsets in multiple sites shown in representative flow cytometry plots (left, D337 and D344), and boxplots of compiled data from 18–55 donors for each site (right). (E) CD57 expression by CD56^bright CD16⁻ (light orange) and CD56^dimCD16⁺ (blue) NK cell subsets shown in representative flow cytometry plots (left, D344 and D345) and compiled data from 13–43 donors (right). See also Figure S1A, right panel. Dots on the boxplots show data collected for each individual donor. ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05. Gut refers to both large and small intestines, as we found similar NK cell and subset frequencies for these two sites (Figures S1B and S1C). BM, bone marrow; LLN, lung-draining lymph node; MLN, mesenteric lymph node.

Figure 2.. NK Cell Subset Distribution in Tissues Is Independent of Age, Sex, and CMV Serostatus

(A) Scatterplots showing frequency of NK cells in blood and tissue sites as a function of age for each individual donor. The line of best fit was determined by Pearson correlation; the Pearson correlation coefficient for each comparison is designated as ‘‘pearsonr’’ in each plot (left). Right: NK cell frequency in different sites in donors stratified by CMV serostatus (CMV⁺, seropositive; CMV⁻, seronegative) and sex (male or female). (B) Scatterplots showing the ratio of CD56^dimCD16⁺:CD56^brightCD16⁻ NK cells in blood and tissue sites as a function of age for each donor, with line of best fit and Pearson coefficient indicated as in (A). See also Figure S1A, middle panel. Right: the ratio of CD56^dimCD16⁺:CD56^brightCD16⁻ NK cells in different sites in donors stratified by CMV serostatus (top) and sex (bottom). (C) Scatterplots showing the distribution of terminally differentiated NK cells (CD56^dimCD16⁺CD57⁺) in blood and tissue sites as a function of age for each donor; the line of best fit and Pearson correlation coefficient indicated in each plot (left). See also Figure S1A, right panel. Right: the distribution of terminally differentiated NK cells in different sites in donors stratified by CMV serostatus (top) and sex (bottom). (D) Representative flow cytometry plots (left) showing the distribution of CMV-responsive (CD56^dimCD16⁺CD57⁺NKG2C⁺) memory-like NK cells in a representative CMV-seronegative (CMV⁻, D344) and CMV-seropositive donor (CMV⁺, D349). Right: boxplots showing compiled distribution of CMV-responsive NK cells from HLA: C1, Bw4/C1, Bw4/C2, and Bw4/C1/C2 CMV⁺ (n = 12–17) and CMV⁻ (n = 9–12) donors, data between the groups was compared using Mann-Whitney U test. See also Figure S2A. Dots on the boxplots show data collected for each individual donor. **p ≤ 0.01; *p ≤ 0.05; ns, non-significant. BM, bone marrow; LLN, lung-draining lymph node; MLN, mesenteric lymph node.

Figure 3.. Tissue Localization Shapes the Functionality of NK Cells

(A) Expression of granzyme B (GzmB) by NK cell subsets in different tissue compartments shown in representative histograms (left, D455 and D456) and graphs of compiled frequency of GzmB⁺ NK cell subsets from 8–19 donors (right). See also Figure S2B. (B) Expression of the CD16 adaptor molecule FcεRIγ by NK cell subsets in different sites shown in representative histograms (left, D344); numbers in each plot represent median fluorescence intensity (MFI) of FcεRIγ for each subset (light orange, CD56^brightCD16⁻; blue, CD56^dimCD16⁺). Boxplots (right) show ∆MFI FcεRIγ (compiled from 10–25 donors) calculated by subtracting the FcεRIγ MFI of control (CD3⁺) cells from the FcεRIγ MFI of each NK cell subset. Comparison of ∆MFI from CD56^dimCD16⁺ cells between LN and gut versus blood, BM, spleen, and lung indicated by red asterisk. (C) IFN-γ secretion following culture of sorted NK cells from indicated sites with cytokines (see STAR Methods) shown as representative histograms (left, D388), and boxplots showing frequency of IFN-γ⁺ NK cells for each site compiled from 4–7 donors (right). (D) CD107a expression following activation of NK cells from indicated sites by co-culture with K562 cells for 4 h (see STAR Methods) shown as representative histograms (left, D454), and boxplots showing frequency of CD107a⁺ NK cells for each site compiled from 8–13 donors (right). Dots on the boxplots show data collected for each individual donor. ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05. BM, bone marrow; LLN, lung-draining lymph node; MLN, mesenteric lymph node.

Figure 4.. Transcriptional Analysis of CD56^brightCD16⁻ and CD56^dimCD16⁺ NK Cells across Sites

(A) PCA plot generated from top 500 differentially expressed (DE) genes between CD56^brightCD16⁻ and CD56^dimCD16⁺ NK cell subsets isolated from 5 sites (blood, BM, spleen, lung, and lung-draining LN). See Table S2 for complete gene list. (B) Heatmap of top 50 globally DE genes between CD56^brightCD16⁻ and CD56^dimCD16⁺ NK cell subsets from all sites. (C) Heatmap showing the top DE transcription factors between CD56^brightCD16⁻ and CD56^dimCD16⁺ NK cells from all sites. (D) PCA plot for CD56^brightCD16⁻ (top) and CD56^dimCD16⁺ (bottom) NK cells isolated from five sites. Each dot in the PCA plots is a separate donor sample, and ellipses around samples indicate 95% confidence ellipses (see STAR Methods). B, blood; M, bone marrow; S, spleen; L, lung; N/LLN, lung-draining lymph node. See also Figures S3 and S4.

Figure 5.. Tissue Residence Profile of CD56^brightCD16⁻ NK Cells

(A) GSEA plot showing the enrichment of core tissue resident-memory T (Trm) cell genes within CD56^brightCD16⁻ NK cells from all tissue sites. See also Figure S5A. (B) Heatmap showing the expression of select core Trm signature genes differentially expressed in tissue CD56^brightCD16⁻ NK cells and not expressed in blood CD56^brightCD16⁻ or CD56^dimCD16⁺ NK cells in all sites. (C) Cell surface expression of canonical tissue residence markers CD69 and CD103 on CD56^brightCD16⁻(light orange) and CD56^dimCD16⁺ (blue) NK cell subsets in different tissue compartments shown in representative flow cytometry plots (top, D337 and D351) and boxplots showing percent CD69⁺NK cells within each subset in different tissue compartments compiled from 18–40 donors. Dots on the boxplots show data collected for each individual donor. See also Figure S5B. ***p ≤ 0.001; **p ≤ 0.01. B, blood; M/BM, bone marrow; S, spleen; L, lung; N/LLN, lung-draining lymph node; MLN, mesenteric lymph node.

Figure 6.. Heterogeneity of Tissue Resident NK Subsets across Sites

(A) Representative SPADE plots of NK cells from blood, BM, spleen, lung, gut, LLN, and MLN stained with tissue-residence markers. The two plots on the left show the distribution NK cells along the maturation axis. The plots on the right show the expression and distribution of select residence markers and marker for terminal differentiation on NK cells along the maturation axis. Solid black arrow identifies CD103- and CD49a-expressing NK cell cluster and dashed black arrow identifies the CXCR6-expressing NK cell cluster. See also Figure S5C. (B) Boxplots showing the distribution of CD69⁺CD103⁺CD49⁺CXCR6⁻ (left) and CD69⁺CD103⁻CD49a⁻CXCR6⁺ (right) CD56^brightCD16⁻, NK cells in indicated tissue sites compiled from 5–10 donors. Dots on the boxplots show data collected for each individual donor. Red asterisk, comparison between lung and tissue site; purple asterisk, comparison between gut and tissue site; orange asterisk, comparison between BM and tissue site; green asterisk, comparison between spleen and tissue site. ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05. BM, bone marrow; LLN, lung-draining lymph node; MLN, mesenteric lymph node.

Figure 7.. High-Dimensional Flow Cytometry Profiling Identifies Tissue-Specific Patterns of NK Development and Maturation

NK cells from blood, bone marrow (BM), spleen, lung, gut, lung-draining LN (LLN), and mesenteric LN (MLN) were stained with the high-dimensional antibody panel specific for multiple NK cell maturation and function markers (see STAR Methods). See also Figure S6. (A) Force-directed plot showing the relationship between different NK cell communities identified by multi-dimensional analysis (data compiled from 5 donors). Each node is an aggregation of similar cells resulting from downsampling of data, and collection of similar colored nodes forms 15 distinct NK cell clusters. Colored text boxes show markers used to differentiate each cluster (1–15). (B) Plots showing the distribution of NK cell communities across the five donors. (C) Heatmap showing the differential expression of cell surface markers used for clustering and community identification in the multi-dimensional analysis in (A). (D) Scatterplots showing distribution of NK cell states in each tissue among the clusters identified by multi-dimensional analysis. For (B) and (D), colored shapes delineate boundaries corresponding to each cluster identified in (A).